In response to RFA-MH-12-140, Development of Tools to Explore the Synaptome, we propose to develop a procedure to trap and isolate recycling synaptic vesicles as an essential first step in analyzing how vesicle protein composition varies with changes in synaptic activity, drug treatment, and under pathological conditions. The ability of synaptic vesicles to undergo multiple rounds of fusion is crucial to maintaining fast, efficient neurotransmission. Synaptic vesicles are formed and recycle via endocytosis. At many synapses, a small proportion of the total vesicle population mediates the majority of neurotransmission while the larger portion remains untapped in a reserve pool. Therefore, proteomic analyses using traditional vesicle purification schemes cannot track changes in the protein composition of the recycling pool. To address this we will develop a scalable, readily applied purification protocol for recycling synaptic vesicles that relies on transiently trapping recycling vesicles as a means of separating them from the total vesicle pool. This approach will open a new field of investigation into the complex endocytotic machinery that regulates vesicle recycling. It will provide a means to test the effects of drugs, genetic manipulations, and pathological conditions on this very important process.